UMASS/AMHERST 


LIBRARY 

OF  THE 

l^ff 

lijBMBl 

r^  wbhwB^v /^r 

^^ 

MASSACHUSETTS 

AGRICULTURAL 

COLLEGE 

NO.__Ll£)  5:8  _____  DATE. 3-tJS51^__ 

souRCE_X'.T._X.jyL.B:uiieTi... 

SF                 ' 

8-^1 

,  P78              1 

i 


|s  book   may   be  kept  out 

TWO   WEEKS 

7,    and    is    subject    to    a     fine     of  TWO 
INTS   a  day   thereafter.      It  will  be  due  on 
day  indicated  below. 


CV.M^ 


Digitized  by  the  Internet  Archive 

in  2010  with  funding  from 

Boston  Library  Consortium  IVIember  Libraries 


http://www.archive.org/details/studyofexperimenOOprud 


A  STUDY  OF 


EXPERIMENTAL  PNEUMONITIS  IN 
THE  RABBIT 


INDUCED  BY   THE  INTRATEAOHEAL  INJECTION   OF 
DEAD   TUBERCLE   BACILLI 


BY 


T.  MITCHELL  PRUDDEN,  M.  D. 

DIRECTOR  OF  THE  PATHOLOGICAX  LABORATORY  OP 
THE  COLLEGE  OF  PHYSICIANS  AND  SURGEONS,  COLUMBIA  COLLEGE,  NEW  YORK 


BEPBINTED  FROM 

THE  NEW  TORE  MEDICAL  JOURNAL 

FOR  DECEMBER  5,  1891 


NEW    YORK 

D.    APPLETON    AND    COMPANY 

1891 


T79 


COPTBIGHT,    1891, 

By  D.   APPLETON  AND  COMPANY. 


A   STUDY   OF 
EXPERIMENTAL  PNEUMONITIS  IN  THE  RABBIT, 

INDUCED  BY  THE 
nSfTRATRACHEAL  INJECTION  OF  DEAD  TUBERCLE  BACILLI. 


Introduction. 


A  GREAT  deal  of  the  morphological  complexity  of  the 
lesions  of  the  lungs  in  both  acute  and  chronic  tuberculosis 
is  due  to  inflammatory  processes  which  do  not  present  the 
characteristic  features  of  tubercular  inflammation.  When 
we  have  taken  account  of  the  miliary  tubercles,  both 
single  and  conglomerated,  of  the  larger  and  smaller  masses 
of  epithelioid-cell  growth  which  we  call  diffuse  tubercle  tis- 
sue, and  of  the  various  aggregates  of  these — often  in  a  con- 
dition of  more  or  less  advanced  coagulation  necrosis  ;  when 
we  have  further  brought  into  line  that  series  of  more  or 
less  extensive  inflammatory  consolidation  of  the  lungs  in 
which,  without  the  development  of  characteristic  tubercle 
tissue,  coagulation  necrosis  and  often  disintegration  of 
both  lung  and  exudate  occur,  under  the  influence  of  the 
living  growing  tubercle  bacillus — there  still  remains  a  series 
of  intra-alveolar  inflammatory  exudations  about  and  among 
the  more  characteristic  tubercular  areas  whose  cause  and 
origin  are  not  sufiiciently  understood.  These  exudations 
are  sometimes  fibrinous,  sometimes  epithelioid  in  character  ; 
sometimes  they  are  largely  composed  of  small  spheroidal 
cells  ;,  or,  which  is  more  frequently  the  case,  all  three  forms 
of  exudate  are  intermingled. 

No  doubt  a  double  infection  sometimes  occurs,  so  that 
associated  with  the  lesions  directly  induced  by  the  tubercle 


4        EXPERIMENTAL  PNEUMONITIS  IN   THE   RABBIT. 

bacillus  are  those  brouglit  about  by  tlie  pneumococcus  or 
the  pyogenic  bacteria,  or  by  other  occasional  inciters  of 
suppurative  inflammation.  But  this  appears  to  be  of  com- 
paratively infrequent  occurrence. 

On  the  other  hand,  there  is  not  infrequently  in  tubercu- 
losis a  considerable  formation  of  new  connective  tissue  in 
the  lungs,  either  circumscribed  or  diffuse,  the  relationship 
of  which  to  the  specific  tubercular  lesion  is  not  very  clear. 

It  was  with  a  view  of  learning,  if  possible,  to  what  ex- 
tent dead  tubercle  bacilli  may  be  capable  of  inducing  these 
and  other  phases  of  complicating  non-specific  forms  of  in- 
flammation of  the  lungs  that  the  studies  were  undertaken 
which  are  now  to  be  recorded. 

Experiments. 
In  a  paper  recently  published  by  Dr.  Hodenpyl  and 
myself  *  it  was  shown  that  dead  tubercle  bacilli,  introduced 
in  moderate  numbers  into  the  ear  vein  of  the  rabbit,  are 
capable  of  inducing  after  a  time  at  their  seat  of  lodgment 
in  the  blood-vess'els,  especially  of  the  lungs  and  liver,  cir- 
cumscribed growths  of  new  cells  which  in  many  respects 
closely  resemble  miliary  tubercles.  These  new  growths 
differ,  however,  fundamentally  from  genuine  miliary  tuber- 
cles in  that  they  do  not,  so  far  as  we  could  observe,  under- 
go cheesy  degeneration  and  never  contain  live  tubercle 
bacilli,  and  are  hence  not  infectious.  Hand  in  hand  with 
the  development  of  these  new  tissue  structures  there  occurs, 
under  the  influence  of  the  dead  tubercle  bacillus,  a  prolifer- 
ation of  endothelium  and  an  extravasation  of  leucocytes. 
It  was  shown,  in  other  words,  that  dead  tubercle  bacilli  pos- 
sess not  only  positive  chemotactic  powers,  but  are,  in  a 
marked  and  peculiar  way,  capable  of  stimulating  various 
phases  of  cell  proliferation.  But,  in  order  to  produce  these 
tubercle-like  structures  in  the  simple  uncomplicated  forms 
described  in  our  paper  above  referred  to,  it  is  necessary,  so 
far  as  our  observations  go,  to  introduce  the  dead  bacilli 
well  distributed  in  very  minute  particles  through  the  fluid 
used  for  injection,  because  in  this  way  one  avoids  any  con- 
siderable immediate  vascular  disturbance  at  the  seat  of  lodg- 
ment of  the  dead  germs. 

*  N.  Y.  Medical  Journal,  June  6  and  20,  1891. 


EXPERIMENTAL   PNEUMONITIS   IN   THE   RABBIT.         5 

If,  on  the  other  hand,  we  inject  into  the  ear  vein  of  the 
rabbit  an  emulsion  of  dead  tubercle  bacilli  in  which,  the 
flocculi  formed  of  the  bacillary  masses  are  large,  so  that  they 
block  up  the  smaller  blood-vessels  where  they  lodge,  one  gets 
an  entirely  different  series  of  results.  This  is  especially 
marked  in  the  lungs,  to  which  organs  our  attention  in  this 
paper  is  largely  limited.  It  will  be  remembered  that  in  our 
former  experiments  the  characteristic  lesions  were  slow  in 
developing,  from  two  to  six  weeks  often  elapsing  before 
any  gross  lesions  were  produced,  while  in  some  cases  no 
gross  but  only  microscopical  changes  appeared.  The  ani- 
mals did  not,  as  a  rule,  experience  any  ill  effects  from  their 
dosage. 

But  when  larger  flocculi  of  dead  bacilli  are  injected  in 
considerable  quantity,  a  certain  proportion  of  the  animals 
die  at  once  from  cerebral  embolism.  Others  fall  directly 
into  convulsions,  apparently  from  the  same  cause,  but  speedi- 
ly recover.  The  larger  proportion,  however,  do  not  appear 
to  suffer  from  the  immediate  effects  of  the  injection,  if  this 
be  made  slowly.  If,  now,  the  animals  which  have  survived 
the  injection  of  these  larger  masses  of  bacilli  be  killed  at 
intervals  of  from  forty-eight  hours  to  ten  days,  or  if  they 
die  during  this  period,  it  will  be  found  in  a  considerable 
proportion  of  cases  that  even  as  soon  as  the  second  day 
after  the  injection  the  lungs  are  more  or  less  thickly  beset 
with  small,  white,  rounded,  or  branching  masses  of  solidified 
lung  tissue. 

Microscopical  examinations  of  these  solid  areas  show 
that  they  consist  of  one  or  more  much-dilated  small  blood- 
vessels surrounded  by  an  irregular  zone  of  air  spaces,  both 
of  which  are  densely  packed  with  small  spheroidal  cells  re- 
sembling leucocytes.  In  sections  stained  for  tubercle  ba- 
cilli it  will  be  seen  that  within  the  blood-vessels,  intermin- 
gled with  the  leucocytes  and  blood-plates  which  form  the 
bulk  of  the  thrombi,  are  large  scattered  masses  of  tubercle 
bacilli.  But  the  bacilli  are  not  confined  to  the  vessels. 
Everywhere  in  the  air- vesicles  of  the  consolidated  areas  they 
lie  scattered  among  the  cells  which  fill  the  air-spaces  or  are 
clustered  within  them.  ,The  whole  picture  conveys  the  im- 
pression that,  either  owing  to  the  disturbance  of  the  circula- 
tion by  the  occluding  bacterial  embolus  or  to  the  irritating 


0        EXPERIMENTAL   PNEUMONITIS   IN   THE   RABBIT. 

effects  of  the  dead  germs  themselves,  there  has  been  not 
only  a  dense  gathering  of  leucocytes  in  the  vessels,  forming 
voluminous  thrombi,  but  that  hand  in  hand  with  the  emi- 
gration of  leucocytes  from  the  affected  vessels  there  has 
been  a  diapedesis  of  dead  tubercle  bacilli.  Whatever  may 
be  the  reason,  the  fact  is  that  within  forty- eight  hours  the 
tubercle  bacilli  in  large  numbers  have  got  outside  the  blood- 
vessels where  they  first  lodged  and  are  mingled  with  the 
exudation  in  the  contiguous  air-vesicles.  While  this  great 
collection  of  leucocytes  in  the  blood-vessels  and  in  their  ad- 
jacent air-vesicles  may  be  in  part  due  to  the  simple  embolic 
vascular  disturbance,  it  would  seem  to  me  that  it  may  be 
largely  owing  to  the  marked  chemotactic  powers  which  the 
dead  tubercle  bacilli  possess,  as  has  been  shown  by  several 
observers. 

I  have  not  followed  this  special  line  of  observation  be- 
yond a  series  of  injections  on  fourteen  rabbits,  because  the 
point  which  I  had  in  view  can  be  more  directly  reached  by 
a  form  of  experiment  which  eliminates  the  extensive  and 
often  profound  vascular  changes  thus  brought  about. 

What  1  wished  to  ascertain  in  these  studies  was  the  ef- 
fects of  dead  tubercle  bacilli  introduced  directly  into  the 
air-vesicles  of  the  lung  of  the  rabbit  in  considerable  quan- 
tity through  the  trachea.  To  record  these  results  is  the 
primary  purpose  of  this  paper. 

The  material  used  for  the  intratracheal  injections  was 
prepared  as  follows :  A  voluminous  flask  culture  of  the  tu- 
bercle bacilli  in  glycerin  bouillon,  two  months  old,  was  fil- 
tered through  sterilized  filter  paper  to  separate  the  bacterial 
mass.  The  latter  was  now  carefully  washed  in  the  filter 
with  large  quantities  of  sterilized  water,  and  then  with 
about  a  hundred  times  its  volume  of  distilled  water  was 
steamed  in  a  small  flask  for  two  hours  in  the  Arnold  steril- 
izer. The  fluid  was  now  filtered  off — to  remove  any  of  the 
metabolic  products  of  the  life  process  of  the  bacillus  which 
might  be  soluble  under  these  conditions — and  again  thor- 
oughly washed  in  the  filter.  The  bacterial  mass,  while  still 
moist,  was  now  again  mixed  with  about  one  hundred  times 
its  volume  of  water  and  again  boiled  for  two  hours,  when 
it  was  ready  for  use. 

I  have,  in  another  set  of  experiments,  boiled  the  cult- 


EKtE  III  MENTAL    PNEUMONITIS   IN    THE   RABBIT.         7 

lives  in  fifty  per  cent,  of  glycerin  for  an  equal  time,  filtering 
off  and  washing  in  the  same  way. 

In  another  set  of  experiments  I  have  used  glycerin 
agar  cultures  of  the  tubercle  bacillus,  sterilized  and  washed 
in  the  same  way. 

While  these  three  sets  of  animal  experiments  were  per- 
formed separately  and  at  different  times,  the  results  were 
identical,  so  that  we  may  consider  them  as  forming  one 
series. 

It  was  now  assumed  that  this  milky  emulsion,  which- 
ever way  prepared,  contained  no  living  tubercle  bacilli,  and 
that  all  the  poisonous  materials  which  might  have  been 
present  in  the  fluids  of  the  original  culture  or  clinging  to 
the  surfaces  of  the  dead  bacilli  had  been  removed  from  the 
material,  at  least  in  so  far  as  they  are  soluble  in  boiling 
water  or  in  glycerin  and  water. 

The  mode  of  operative  procedure  was  as  follows :  After 
cutting  away  the  hair  and  sterilizing  the  skin  about  the 
neck  of  the  rabbit,  a  small  incision  was  made,  exposing  the 
trachea,  and  the  needle  of  the  injecting  syringe  was  thrust 
through  its  wall.  About  two  cubic  centimetres  of  a  milky 
emulsion  of  dead  tubercle  bacilli  was  slowly  introduced  in 
this  way,  the  animals  being  held  upright  and  turned  about 
from  side  to  side  during  and  for  a  few  moments  after  the 
injection,  so  that  the  fluid  might  run  well  down  into  various 
parts  of  the  lungs.  The  animals  bear  this  injection  per- 
fectly well,  often  experiencing  a  momentary  dyspnoea,  and 
the  wounds  of  the  neck  always  closed  promptly. 

Thirty-four  animals  were  operated  upon  in  this  way  and 
were  killed  at  the  following  intervals  :  Two  on  the  first  day 
after  the  operation,  three  on  the  second  day,  one  on  the 
fourth,  one  on  the  fifth,  two  on  the  sixth,  one  on  the  eighth, 
one  on  the  eleventh,  one  on  the  thirteenth,  one  on  the  seven- 
teenth, one  on  the  eighteenth,  two  on  the  twentieth,  three 
on  the  twenty-first,  two  on  the  twenty-third,  one  on  the 
twenty-fourth,  two  on  the  twenty-seventh,  two  on  the  twenty- 
ninth,  two  on  the  thirty-first,  one  on  the  thirty-fourth,  three 
on  the  forty-first,  one  on  the  fifty-third,  and  one  on  the 
seventy-second. 

The  results  of  the  introduction  of  dead  tubercle  bacilli 
into   the  lungs  of  rabbits  through  the  trachea  in  this  way 


8        EXPERIMENTAL   PNEUMONITIS   IN   THE   RABBIT. 

are  so  certain,  so  positive,  and  so  uniform,  tliat  it  seems  to 
me  wiser  to  give  the  general  effects  of  the  action  of  the 
dead  germs  than  the  story  of  particular  animals. 

The  gross  appearance  of  the  lungs  of  animals  operated 
upon  in  this  way  changes  considerably  from  day  to  day, 
and  the  general  topography  of  the  lesions  varies  greatly 
with. the  vicissitudes  of  the  experiment,  in  accordance  with 
which  the  masses  of  bacilli  may  be  abundant  or  scanty, 
large  or  small,  are  grouped  in  one  lung  or  part  of  a  lung,  or 
are  widely  disseminated  through  both  organs.  Thus,  one 
or  both  lungs  may  be  irregularly  besprinkled  with  small 
miliary  or  submiliary  areas  of  consolidation,  or  such  areas 
may  be  associated  with  larger  areas  of  diffuse  consolidation 
which  may  occupy  portions  of  a  lobe,  or  a  whole  lobe,  or 
nearly  a  whole  lung.  The  situation  of  the  lesion  is  directly 
dependent  iipon  the  seat  of  lodgment  of  the  dead  bacilli, 
while  its  extent  appears  to  be  directly  and  uniformly  de- 
pendent upon  the  amount  of  bacilli  introduced  or  the  length 
of  time  allowed  to  elapse  between  the  injection  and  the  kill- 
ing of  the  animals.  No  lesions,  either  gross  or  microscopi- 
cal, were  found  in  any  of  the  viscera  except  the  lungs. 

The  general  course  of  events  after  the  introduction  of 
dead  tubercle  bacilli  into  the  lungs  of  rabbits  under  the 
conditions  set  forth  above  is  as  follows  : 

As  early  as  twenty-four  hours,  if  the  animal  is  killed, 
extensive  lesions  are  developed.  The  lungs  are  moderately 
congested,  and  namerous  white  spots  of  consolidation,  from 
0*5  to  2  millimetres  in  diameter,  are  seen  shimmering 
through  the  pleura.  The  cut  surface  of  the  lungs,  as  a 
rule,  reveals  more  extensive  lesion  than  would  be  expected 
from  the  superficial  inspection  of  the  organs.  The  cut  sur- 
faces usually  show  large  numbers  of  irregularly  branching 
or  isolated  and  scattered  white,  dense,  airless  areas,  com- 
monly most  abundant  posteriorly,  which  correspond  with 
the  bronchi  and  groups  of  their  adjacent  air-spaces.  There 
is  usually  special  congestion  of  the  blood-vessels  imme- 
diately about  these  consolidated  areas  in  some  parts  of  the 
lungs. 

The  gross  appearance  of  the  cut  surface  of  a  lung 
twenty-four  hours  after  the  injection  is  indicated  by  Fig.  1. 

The  microscopical    examination   shows  that  the  white 


EXPERIMENTAL    PNEUMONITIS   IN   THE    RABBIT.         9 

areas  of  consolidation  are  due  to  an  extreme  filling  and  dis- 
tention of  tlie  smaller  bronchi  and  portions  of  their  con- 
tio-tious  air-spaces  with  densely  packed  masses  of  small 
spheroidal  cells  resembing  leucocytes.  Many  of  the  smaller 
blood-vessels  in  these  consolidated  areas  are  packed  with 
small  spheroidal  cells.  In  some  places  there  is  a  very  slight 
increase  in  the  number  of  the  epithelial  cells  of  the  air- 
vesicles. 

Sections  stained  for  tubercle  bacilli  show  that  wherever 
this  gathering  of  small  spheroidal  cells  had  occurred  there 
are  tubercle  bacilli,  usually  in  very  large  numbers.  Indeed, 
tubercle  bacilli  are  nowhere  found  in  the  air-spaces  in  any 
considerable  numbers  without  being  closely  intermingled 
with  these  small  cells. 

While  the  medium  and  smaller  bronchi  in  the  consoli- 
dated areas  are  closely  packed  with  the  small  spheroidal 
cells  and  dead  tubercle  bacilli,  the  epithelial  lining  of  the 
bronchi  is  almost  wholly  intact.  There  are  neither  inflam- 
matory nor  degenerative  nor  exfoliative  changes  in  the 
bronchial  epithelium.  It  appears  more  as  if  the  small  cells 
had  been  forced  up  into  the  bronchi  from  the  groups  of 
communicating  air-spaces  which  are  so  densely  packed  with 
them.  In  some  places,  especially  where  the  accumulation 
of  small  spheroidal  cells  in  the  air-spaces  is  large  and  the 
dead  bacilli  are  numerous,  the  cell  bodies  appear  homo- 
geneous and  their  outlines  indistinct ;  but  their  nuclei  are, 
as  a  rule,  well  stained,  so  that  the  dense  masses  of  exudate 
present  somewhat  the  appearance  of  coagulation  necrosis, 
but  are  still  evidently  not  in  this  condition. 

The  result,  then,  of  the  action  of  dead  tubercle  bacilli 
in  large  numbers  in  the  air-spaces  of  the  rabbit's  lung  for 
twenty-four  hours  is,  in  general,  the  accumulation  in  enor- 
mous numbers  about  the  bacilli,  and  nowhere  else,  of  small 
spheroidal  cells  resembling  leucocytes. 

The  microscopical  appearance  of  a  small  bronchus  show- 
ing bacilli  is  given  in  Fig.  2,  and  of  two  adjacent  air-vesi- 
cles in  Fig.  3. 

At  the  end  of  the  second  day  the  gross  appearance  of 
the  lungs  differs  little  from  that  at  the  end  of  the  first.  Micro- 
scopical examination  shows  that  the  small  bronchi  and  air- 
spaces in  groups  are   densely  packed  and  distended  with 


10      EXPERIMENTAL   PNEUMONITIS   IN   THE    RABBIT. 

small  spheroidal  cells,  many  of  wMch,  especially  where  there 
are  many  tubercle  bacilli,  have  undergone  a  peculiar  change. 
The  nuclei  remain  apparently  unaltered,  but  the  bodies  are 
swollen,  no  longer  granular,  but  nearly  homogeneous  and 
glassy  in  appearance,  and  in  places  have  run  together,  form- 
ing shining  masses.  This  change  in  the  accumulated  cells 
may  begin,  as  already  indicated,  and  may  even  be  fairly 
well  marked  within  twenty-four  hours  after  the  injection. 

The  empty  air-spaces  close  to  those  which  are  thus  filled 
with  small  cells  and  dead  tubercle  bacilli  are  so  squeezed 
that  their  lumina  are  for  the  most  part  closed.  They  then 
form  an  ill-defined,  irregular,  solid  zone  of  atelectasis  im- 
mediately about  and  in  places  running  into  the  exudative 
area. 

In  animals  killed  from  the  fourth  to  the  sixth  day  after 
the  injection  into  the  trachea  the  gross  appearance  of  the 
lungs  is  essentially  similar  to  that  presented  at  the  end  of 
the  first,  save  that  around  the  white  consolidated  areas  there 
are  usually  visible  narrow,  irregular,  grayish,  translucent 
zones  of  consolidation. 

The  smaller  bronchi,  in  places,  and  their  groups  of  re- 
lated air-spaces,  are  filled,  as  at  an  earlier  period,  with  dense 
masses  of  intermingled  small  spheroidal  cells  and  dead  but 
readily  stained  tubercle  bacilli.  Where  these  central  masses 
are  dense  and  harbor  many  bacilli,  the  cells  are,  as  at  an 
earlier  period,  homogeneous  and  diffusely  outlined.  The 
blood-vessels  in  and  near  these  consolidated  areas  are  often 
crowded  with  small  spheroidal  cells,  and  around  many  of  the 
larger  vessels  of  these  areas  there  is  a  perivascular  sheath 
of  spheroidal  cells. 

The  translucent  consolidated  borders  of  these  areas  are 
largely  formed  by  the  filling  of  the  surrounding  air-spaces 
with  collections  of  epithelioid  cells,  or  giant  cells,  or  small 
spheroidal  cells,  or  all  of  these  variously  intermingled. 
There  is  much  variability  in  the  number  of  giant  cells  in 
the  new  tissue,  which  I  can  only  account  for  by  individual 
peculiarity  of  the  animals.  In  an  animal  killed  on  the  fourth 
day,  for  example,  there  was  almost  no  tendency  to  produce 
giant  cells,  while  on  the  fifth  day  an  animal  treated  similarly 
showed  them  in  enormous  numbers. 

Tubercle  bacilli   are  present  in   this   border  zone,   but 


EXPERIMENTAL   PNEUMONITIS   IN   THE   RABBIT.      H 

in  less  numbers  than  in  the  primary  areas  of  consolida- 
tion. 

Fig.  4  shows  the  edge  of  one  of  these  areas  of  consoli- 
dation from  the  lung  of  a  rabbit  killed  four  days  after  the 
tracheal  injection  of  the  dead  tubercle  bacilli. 

The  essential  change,  then,  which  takes  place  in  the 
lesion  toward  the  end  of  the  first  week  is  the  formation  and 
accumulation  of  cells — epithelioid  and  giant — in  the  air- 
spaces about  the  primary  focus  of  small-cell  collection,  and 
the  infiltration  and  thickening  of  the  walls  of  the  involved 
air-spaces. 

In  the  second  week  two  distinct  sets  of  changes  occur  in 
the  consolidated  areas,  whether  these  be  large  or  small : 

First,  there  is  a  well-marked  tendency  to  disintegration 
and  absorption  in  the  central  portion  of  the  solid  areas — 
that  is,  in  that  portion  first  to  appear  at  the  seat  of  lodg- 
ment of  the  bacilli,  and  which  is  formed  of  densely  packed 
masses  of  small  spheroidal  cells  mingled  with  large  num- 
bers of  dead  bacilli.  The  walls  of  the  old  air-spaces  in  this 
area  are  apparently  dead,  so  that  the  central  portion  of  the 
solid  areas  is  made  up  of  a  mass  of  necrotic,  disintegrat- 
ing tissue,  which  appears  to  be  growing  smaller  by  absorp- 
tion. 

The  second  set  of  changes  is  in  the  translucent  periph- 
eral zone  which  now  forms  the  most  prominent  feature  of 
the  consolidated  area.  This  peripheral  translucent  zone  is 
formed  of  air-spaces  more  or  less  closely  packed  with  epi- 
thelioid cells  and  giant  cells,  the  former  largely  prepon- 
derating and  showing  in  the  most  exquisite  w'ay  the  varied 
nuclear  figures  of  indirect  cell  division.  The  walls  of  the 
air  spaces  of  this  peripheral  zone  are  thickened,  apparently 
from  the  accumulation  of  fiuid  and  small  spheroidal  cells 
within  them,  and  their  blood-vessels  are  in  places  widely 
distended  with  leucocytes,  so  that  the  areas  of  consolida- 
tion are  often  distinctly  bordered  by  a  rim  of  small  sphe- 
roidal cells. 

In  the  immediate  vicinity  of  clusters  of  the  dead  tu- 
bercle bacilli  the  formation  of  epithelioid  cells  is  not  so 
regular  and  perfect  as  at  a  little  distance  from  them,  or 
where  they  are  more  sparsely  scattered. 

Near  the  clusters  of  bacilli  the  new  cells  are  massed  in 


12      EXPERIMENTAL   PNEUMONITIS   IN   THE   KABBIT. 

the  form  of  ill- defined  giant  cells,  or  the  bacteria  are  sur- 
rounded by  an  irregular  granular  or  translucent  material, 
into  which,  here  and  there,  leucocytes  have  penetrated.  One 
not  infrequently  sees  that  the  epithelioid  cells  which  have 
developed  around  a  clump  of  dead  bacilli  in  an  air-space 
are  radially  placed  around  it  with  their  nuclei  uniformly 
crowded  to  the  distal  portion  of  the  cell. 

The  result  of  these  minute  alterations  is  that  to  the 
naked  eye  the  solid  areas  at  this  time  present  the  appear- 
ance of  gray,  translucent,  irregular  masses  of  new  tissue, 
having  proportionately  small  and  inconspicuous  white  cen- 
ters. The  general  appearance  of  the  solid  areas  toward  the 
end  of  the  second  week  is  represented,  somewhat  schemati- 
cally, in  Fig.  5. 

In  the  third  week  there  is  a  steady  disappearance  of  the 
necrotic  central  portion  of  the  solidified  areas,  while  in  the 
peripheral  translucent  zone  the  old  air-spaces  are  steadily 
obliterated  by  the  thickening  of  their  walls  and  the  disap- 
pearance of  lines  of  demarkation  between  these  and  the  cell- 
filled  air-spaces.  So  that  the  original  air-spaces  come  to  be 
represented  by  larger  and  smaller  collections  of  closely 
packed  epithelioid  cells  lying  in  the  meshes  of  a  very  vascu- 
lar and  very  cellular  new  connective  tissue.  The  blood- 
vessels in  the  new  connective  tissue  have  lost  all  the  topo- 
graphical characters  of  the  original  blood-vessels  of  the 
affected  region,  but  may  be,  in  part,  some  of  these  which 
have  persisted.  On  the  other  hand,  many  of  them  are  cer- 
tainly newly  formed,  since  one  can  find  in  these  sections  the 
various  phases  of  blood-vessel  development,  such  as  are  seen 
in  typical  granulation  tissue.  The  appearance  of  a  typical 
portion  of  this  peripheral  zone  at  about  the  middle  of  the 
third  week  is  shown  in  Fig.  6. 

From  the  third  week  on  the  story  is  usually  one  of  the 
steady  disappearance  of  the  necrotic  center  and  the  con- 
version of  the  peripheral  areas  of  consolidation  into  masses 
of  connective  tissue  with  their  continuous  decrease  in  size. 
The  tubercle  bacilli,  too,  become  steadily  less  abundant. 
This  new  connective  tissue  is  at  first  very  cellular,  as  shown 
in  Fig.  7,  but  the  intercellular  substance  continually  in- 
creases in  amount. 

In  many  of  the  nodules  of  new  connective  tissue  por- 


EXPERIMENTAL    PNEUMONITIS   IN    THE    RABBIT.      13 

tions  of  the  old  air-spaces  are  inclosed  by  the  new  tissue 
and  wholly  separated  from  surrounding  air-spaces  or  con- 
nected with  them  by  flattened  strings  of  epithelioid  cells. 
These  inclosed  air-spaces  are,  as  a  rule,  lined  with  a  con- 
tinuous layer  of  cuboidal  or  flattened  cells.  There  has  ob- 
viously been  a  reversion  of  the  epithelial  cells  lining  the 
inclosed  and  isolated  air-spaces  to  the  embryonal  type,  as 
there  is  under  somewhat  similar  conditions  in  some  phases 
of  chronic  phthisis.  Fig.  S  shows  a  typical  portion  of  one 
of  these  connective-tissue  nodules  at  this  time. 

At  last  nothing  is  left  of  the  consolidated  areas  but 
larger  or  smaller  masses  of  cicatrical  tissue  which  may  or 
may  not  harbor  a  few  still  stainable  granular  tubercle 
bacilli.  The  time  which  is  required  for  the  conversion  of 
the  nodules  produced  in  the  lungs  by  the  injection  of  dead 
tubercle  bacilli  into  dense  cicatrices  varies  considerably, 
depending  upon  the  amount  of  lung  tissue  involved — that 
is,  upon  the  size  of  the  primary  mass.  The  smaller  ones 
may  disappear  to  the  naked  eye  inspection  as  early  as  the 
end  of  the  third  week ;  the  larger  may  remain  for  many 
weeks.  When  whole  lobes  are  involved,  many  vv^eeks  may 
elapse  before  the  conversion  of  the  involved  portion  into 
cicatricial  tissue.  But,  in  general,  the  description  which  I 
have  given  above  expresses  as  closely  as  is  possible,  with 
the  material  at  my  disposal,  the  course  of  events.  Sometimes 
there  appears  to  be  little  tendency  in  many  of  the  small 
nodules  to  the  formation  of  connective  tissue,  but  the  nodules 
consist,  after  the  lapse  of  many  weeks,  of  a  congeries  of  air- 
vesicles  greatly  diminished  in  size  by  the  thickening  of 
their  walls  and  densely  packed  with  epithelioid  cells  and 
giant  cells.  In  one  animal  killed  on  the  twenty-third  day 
extensive  calcification  of  one  of  the  connective-tissu 
nodules  had  occurred. 

I  could  not  observe  that  the  size  or  nutritive  condition 
of  the  animals  made  any  difference  in  the  rate  of  progress  of 
the  changes  in  the  lesions.  As  has  been  already  stated,  the 
lesions  in  the  lungs,  even  vfhen  a  large  part  of  a  lobe  was 
involved,  did  not  appear  to  exert  any  harmful  influence  on 
the  health  and  bearing  of  the  animals. 

Summary. — These  studies  show  that  when  dead  tuber- 
cle  bacilli  are  introduced    in   small    flocculi    into   the  air- 


14      EXPERIMENTAL   PNEUMONITIS   IN   THE   RABBIT. 

spaces  of  the  rabbit's  lung  there  occurs  at  their  seat  of 
lodgment,  first,  a  large  accumulation  of  small  spheroidal 
cells  in  the  air-spaces.  This  is  immediately  followed  by  a 
proliferation  of  epithelioid  cells  and  formation  of  giant 
cells  in  the  contiguous  air-spaces.  Then  occur  gradual 
necrosis,  disintegration,  and  absorption  of  the  primary 
small-celled  center  and  a  conversion  of  the  peripheral  zone 
into  very  cellular  and  vascular  new  connective  tissue. 
Hand  in  hand  with  the  absorption  of  the  necrotic  center 
the  new-formed  connective  tissue  becomes  denser  and  less 
abundant,  until  finally  the  seat  of  lesion  is  indicated  only 
by  a  shred  or  patch  of  dense  connective  tissue,  which,  if 
the  original  lesion  was  not  extensive,  may  be  wholly  in- 
visible to  the  naked  eye.  Sometimes,  however,  but  little  con- 
nective tissue  is  formed  except  in  the  walls  of  the  involved 
air-spaces,  but  the  nodules  persist  for  long  periods  as  a  con- 
geries of  densely  packed  epithelioid  and  giant-cell  masses. 

Remarks. — This  is  a  simple  biological  study,  showing 
the  reaction  of  certain  cells  in  the  rabbit's  lungs  in  the  pres- 
ence of  the  dead  bodies  of  a  well-defined  and  important 
species  of  pathogenic  bacteria.  It  has  no  necessary  bear- 
ing on  human  tuberculosis,  nor  even  on  tubercular  inflam- 
mation in  rabbits.  And  yet  when  we  consider  the  close 
analogy  between  tuberculosis  in  man  and  in  the  rabbit,  and 
the  similarity  between  certain  phases  of  the  lesions  pro- 
duced in  the  rabbit  by  the  living  and  the  dead  germs,  we 
are,  it  seems  to  me,  justified  in  certain  limited  conjectures 
as  to  the  possible  or  probable  bearings  of  such  a  study  on 
our  conception  of  human  pulmonary  tuberculosis. 

We  may,  I  think,  assume  that  of  all  the  tubercle  bacilli 
which  are  present  in  the  body  at  a  given  time  in  any  form, 
of  tuberculosis,  only  a  certain  proportion  are  alive.  This 
we  may  fairly  assume  from  what  we  know  of  the  life 
history  of  the  tubercle  bacillus  under  artificial  cultivation, 
together  with  what  we  know  of  the  life  history  of  bacteria 
in  general,  both  within  and  without  the  body.  New  indi- 
viduals form  and  old  ones  die  with  varying  degrees  of  ra- 
pidity so  long  as  the  environment  favors  vegetative  activity. 
With  the  formation  of  spores  and  the  maintenance  of  the 
life  of  the  species  in  this  way  when  vegetative  activity 
ceases,  we  have  here  nothing  to  do. 


EXPERIMENTAL   PNEUMONITIS   IN   THE   RABBIT.      15 

It  lias  been  abundantly  shown  in  this  study  and  in 
those  on  the  same  theme  which  have  preceded  it  that  the 
power  of  taking  and  retaining  the  characteristic  stain  may 
be  retained  by  the  tubercle  bacillus  long  after  life  in  the 
germ  has  ceased  and  after  its  subjection  to  prolonged  boil- 
ing and  to  the  influence  of  living  body  cells.  It  has  been 
shown,  too,  that  dead  tubercle  bacilli  slowly  disintegrate 
and  finally  disappear  when  surrounded  by  living  body  cells 
and  by  the  body  juices. 

The  primary  difference  between  the  action  of  the  dead 
and  that  of  the  living  tubercle  bacillus  in  the  rabbit  appears 
to  be  that  the  living  bacillus  proliferates  in  the  body  and  pro- 
duces progressive  lesions  with  a  marked  and  characteristic 
tendency  to  generalization  and  to  coagulation  necrosis — de- 
veloping an  acute  infectious  disease  ;  while  the  dead,  bacil- 
lus produces  lesions  closely  similar  morphologically  in  many 
respects,  and  yet  which  are  not  indefinitely  progressive  and 
do  not  tend  to  generalization  or  to  the  production  of  an 
advancing  coagulation  necrosis,  and,  furthermore,  do  not 
induce  an  acute  infectious  disease.  The  necrosis  which  de- 
velops under  the  influence  of  the  dead  germs  differs  from 
that  of  genuine  tuberculosis,  not  only  in  its  morphological 
characters,  but  also  in  the  time  of  its  occurrence.  It  is  one 
of  the  earliest  of  the  changes  following  the  primary  gather- 
ing of  cells  about  the  dead  bacilli  and  attains  at  once  its 
maximum  development.  The  cheesy  degeneration  of  genu- 
ine tuberculosis,  on  the  other  hand,  is  a  gradually  developed 
and  progressive  process,  and  represents  the  usual  culmina- 
tion of  the  lesion. 

We  now  know  that  dead  tubercle  bacilli  can  induce  in 
the  living  body  the  development  of  cell  structures  which, 
within  the  limits  above  indicated,  are  morphologically  char- 
acteristic of  the  lesions  of  tubercular  inflammation.  We  do 
not  know  whether  the  living  tubercle  bacilli  are  capable 
of  stimulating  the  body  cells  to  the  development  of  such 
lesions  or  not,  because  presumably  both  the  living  and  the 
dead  germs  are  present  in  the  ordinary  tubercular  foci.  We 
must  admit  the  possibility  that  in  acute  and  in  chronic  phthi- 
sis a  certain  proportion  of  the  inflammatory  foci  in  the  lungs 
— certain  of  the  broncho-pneumonic  areas — may  be  caused  by 


16      EXPERIMEXTAL   PNEUMONITIS   IN   THE   RABBIT. 

dead  tubercle  bacilli  transported  from  cavities  to  other  parts 
of  the  lungs. 

The  conjecture  was  expressed  in  a  previous  paper  on  the 
effects  of  dead  tubercle  bacilli  by  Dr.  Hodenpyl  and  the 
writer,  that  the  stimulus  to  cell  proliferation  in  tubercular 
inflammation  might  be  due  to  the  bacterio-protein  of  the 
tubercle  bacilli  set  free  during  their  disintegration.  That 
conjecture  this  study  would  seem  to  sustain. 

Whether  the  characteristic  cheesy  degeneration  of  tu- 
bercular inflammation  is  due  to  an  eliminated  metabolic 
product  of  the  living  growing  germ  or  to  some  product  or 
influence  as  yet  wholly  unknown,  remains  to  be  found  out. 
Experimental  studies  in  that  direction  are  in  progress  in  this 
laboratory.  Studies  on  the  effects  of  combining  the  two 
factor's — the  dead  germ  and  the  metabolic  products — by  a 
separate  administration  in  the  same  animal  are  also  under 
way,  but  are  not  yet  sufficiently  advanced  to  permit  of  defi- 
nite conclusions. 

In  view  of  what  we  know  of  the  importance  of  the  mus- 
tering of  various  forms  of  cells  in  the  neighborhood  of  bac- 
terial invaders  of  the  body  as  a  direct  or  indirect  protection 
against  their  ravages,  this  power  of  dead  tubercle  bacilli  to 
stimulate  the  reproduction  of  cells  becomes  of  a  great  deal 
of  apparent  significance. 

I  have  alluded,  in  the  papers  on  this  subject  above  re- 
ferred to,  to  the  possibility  that  the  development  of  tubercle 
tissue  in  the  body  may  be  a  conservative  action  of  great  im- 
portance. The  studies  here  recorded  would  widen  the  scope 
of  that  conjecture  by  suggesting  the  possibility  that  not  only 
the  specific  tubercle  tissue  itself,  but  the  other  cell  accumuT 
lations  which  in  the  lungs  so  often  accompany  it,  may  serve 
an  analogous  purpose  ;  for  we  should  not  forget  that  what- 
ever shall  be  the  ultimate  outcome  of  researches  on  the  ab- 
solute or  relative  importance  of  phagocytosis  and  the  germi- 
cidal power  of  the  body  fluids,  the  efficiency  of  the  latter  is 
ultimately  dependent  upon  cell  activity,  as  Buchner  and 
others  have  repeatedly  insisted. 

In  view  of  this  condition  of  affairs,  it  is  perfectly  possi- 
ble, as  Buchner  *  suggested  very  soon  after  Koch's  first  an- 
nouncement of  the  powers  of  tuberculin,  that  that  substance 

*  Buchner.     Ifiinchener  med.  Wochenschrift,  1890,  No.  47. 


EXPERIMENTAL   PNEUMONITIS   IN    THE   RABBIT.      17 

may  owe  some  of  its  virtues,  if  sucli  it  possesses,  to  proteins 
which  have  resulted  from  the  degeneration  in  the  cultures 
of  the  bodies  of  the  tubercle  bacilli. 

It  is  evident  from  this  study  that  certain  of  the  non- 
characteristic  complicating  lesions  of  the  lungs  in  acute  and 
chronic  phthisis  referred  to  at  the  commencement  of  this 
paper,  leucocytic  and  epithelial  cell  collections  in  the  air- 
spaces of  the  lungs,  as  well  as  the  development  of  new  in- 
terstitial connective  tissue,  may  be  caused  by  the  presence 
of  dead  tubercle  bacilli  at  the  seat  of  lesion. 

We  have  in  this  form  of  experimentation  on  the  rabbit 
a  means  of  study  of  pulmonary  inflammation  wholly  within 
our  control  and  most  useful  for  the  examination  and  dem- 
onstration of  those  forms  of  cell  activity  which  are  involved 
in  indirect  division. 

Finally,  the  power  of  inducing  at  will,  without  seriously 
compromising  the  health  of  the  animal,  small  circumscribed 
foci  of  inflammation  of  varying  intensity,  which  have  a 
definite  history  and  outcome,  may  be  of  no  inconsiderable 
importance  in  the  study  of  the  action  of  drugs  along  the 
lines  suggested  by  Koch's  announcement  of  the  localized 
eifects  of  tuberculin  in  the  body  under  conditions  of  focal 
inflammation  analogous  with  those  here  experimentally  in- 
duced. 

Our  knowledge  of  the  efi^ects  of  the  tubercle  bacillus  in 
the  body  is  now,  I  think,  suflSciently  advanced  for  us  to 
make  at  least  a  conjectural  analysis  of  its  action. 

1.  So  far  as  the  primary  morphological  lesion  is  con- 
cerned, we  may  conjecture  that  the  cell  growth  which  is 
characteristic  of  miliary  tubercle  and  diffuse  tubercle  tissue 
may  be  due  to  the  action  of  the  protein  of  the  bodies  of  the 
germs  set  free  as  they  degenerate  in  contact  with  living- 
cells  ;  and  that  this  production  of  new  tissue  may  not  be 
intrinsically  of  such  practical  significance  as  has  been 
hitherto  supposed ;  or,  if  significant,  it  may  be  so  as  a 
conservative  and  not  as  a  harmful  process. 

2.  It  would  seem  from  our  experiments  that  the  cheesy 
degeneration  which  is  so  constant  an  accompaniment  of  tu- 
bercular lesions  may  be  due  to  some  metabolic  product  of 
the  growth  of  the  tubercle  bacillus  wholly  distinct  from  the 
cell- stimulating  bacterio-protein. 


18      EXPERIMENTAL   PNEUMONITIS   IN   THE   RABBIT. 

3.  There  still  remains  tlie  possibility,  most  evident  on 
clinical  gronnds,  that  beyond  the  factor  which  causes  the 
tissue  cells  to  grow,  and  beyond  the  factor  which  induces 
necrosis,  there  may  be  yet  a  third  agency  of  toxic  nature  to 
which  many  of  the  graver  systemic  effects  of  tubercular  in- 
fection are  due. 

This  mere  suggestion  of  an  analysis  of  the  action  of  the 
tubercle  bacillus  in  the  body  is  made  in  conclusion  here 
only  in  a  tentative  way,  with  a  view  of  affording  a  perhaps 
only  temporary  guiding  thought  in  our  future  study  of  this 
most  important  disease  and  our  future  attempts  to  stay  its 
ravages. 


*  1  ,S  I^ 

=«  'o  o 

O     rt    .„-  ^ 


^vAilk^ 


fro    'bW        W  >f      \ 


t^     m  If    \/^ 


f""^ 


"^ 


(of 


i.  ..■ 


.-.3.      •5^'.-*'^*ia3^ 


^liT  "^ 


■y.  (r 


,4^ 


;i: 


V    A 


\'--,  «         „ 

=   j3   'S 

'"    ,*■     •'•              rB 

>-.  H  -^ 

^  -.  '^'   i 

a        ^ 

'■^'-^-'    fc'Vv 

^  s  ^ 

\l      0 

"*^  'S    ^ 

"5    ^.  f    w 

1  J| 

,v*     *? 

.  •',     t- 

P    'o     ^ 

"^    B~       -"-^^o      -- 


;/    'o   '^   '^     p., 


■/05:'r;,r'->^j  .^  '-,.,;■ 


2  -3   S   S 


'^^    "^  %,  _Av^^a  ^    S 


^  1^^^^*^#^*^i^^-^   <i^sy© 


2     =^ 


.//  "'  ^-. 


m 


5  tS  g  -  CS 

0  a  ^  -S  -^ 
r^  ^  ay  S  a 

1  c  «  a  -7-, 


f=H      V      ^        !^      r^ 


